Finally, Rho proteins and their regulators have been implicated in mediating Selleck Everolimus repulsive guidance signaling (Derijck

et al., 2010; Govek et al., 2005; Hall and Lalli, 2010). Links between Rho GTPase signaling and Sema-plexin-mediated guidance prompted us to examine interactions between Drosophila RhoGEFs, RhoGAPs, and receptor-type guidance molecules. We identified pebble (Pbl), a RhoGEF for Rho1, and RhoGAPp190 (p190), a RhoGAP for Rho1, as signaling molecules with the potential to function downstream of Sema-1a reverse signaling in neurons. Our genetic analyses suggest that Pbl and p190 play key opposing roles in Sema-1a reverse signaling. To investigate links between Rho GTPase regulators and semaphorin/plexin-mediated neuronal guidance, we screened several RhoGEF and RhoGAP proteins for their ability to interact with Drosophila PlexA, PlexB, and Sema-1a in Drosophila S2R+

cells in vitro. We found that Pbl weakly interacts with PlexA, while p190 weakly interacts with both PlexA and PlexB ( Figures 1A and 1B). However, when we performed these same protein interaction assays using the PlexA ligand Sema-1a, we found that both Pbl and p190 proteins Venetoclax concentration robustly interact with Sema-1a, to a much greater degree than with either PlexA or PlexB ( Figures 1A and 1B). These strong interactions are apparently specific since other transmembrane proteins, including Drosophila Off-track (Otk), do not coimmunoprecipitate with either Pbl or p190 when coexpressed in S2R+ cells in these same experiments ( Figures 1A and 1B). We also observed in coimmunoprecipitation (Co-IP) experiments

that neuronally expressed embryonic HA-Pbl and HA-p190 robustly bind to endogenous Sema-1a in vivo ( Figure S1A available online). These observations suggest that Pbl and p190 participate in intracellular signaling cascades downstream of Sema-1a ( Figure 2E). To further characterize the specificity of these interactions between Sema-1a and Pbl, we mapped the regions of Pbl responsible MycoClean Mycoplasma Removal Kit for interactions with Sema-1a, revealing that the N-terminal domain (NTD), which encompasses two tandem BRCT (BRCA1 C-terminal) domains, is necessary and sufficient for mediating Sema-1a binding (Figure S1B). Through a systematic deletion and mutagenesis analysis of the Sema-1a intracellular domain (ICD), we found that Sema-1a ICD[Δ31–60], in which ICD amino acid residues 31–60 are deleted, and ICD[36G/52A] exhibited differential binding properties to full-length p190 and truncated NTD[Pbl] (Figure 1C; highlighted in red). To address whether this difference is due to the absence of the Pbl C-terminal domain (CTD), we next tested the ability of full-length Pbl and p190 to bind to these mutant forms of the Sema-1a ICD; we observed a significant reduction in Pbl binding to both ICD[Δ31–60] and ICD[36G/52A] (Figure 1D).